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Home , Brachiopod, Novocrania anomala, Spiralia

Gąsiorowski and Hejnol EvoDevo (2019) 10:1 https://doi.org/10.1186/s13227-018-0114-1 EvoDevo

RESEARCH Open Access expression in postmetamorphic juveniles of the Terebratalia transversa Ludwik Gąsiorowski and Andreas Hejnol*

Abstract Background: Hox genes encode a family of homeodomain containing transcription factors that are clustered together on chromosomes of many . Some bilaterian lineages express these genes during embryogenesis in spatial and/or temporal according to their arrangement in the cluster, a phenomenon referred to as collinear- ity. Expression of Hox genes is well studied during embryonic and larval development of numerous ; however, relatively few studies focus on the comparison of pre- and postmetamorphic expression of Hox genes in with biphasic cycle. Recently, the expression of Hox genes was described for and larvae of Terebratalia transversa, a rhynchonelliformean brachiopod, which possesses distinct from planktonic larvae to sessile juveniles. During premetamorphic development, T. transversa does not exhibit spatial collinearity and several of its Hox genes are recruited for the morphogenesis of novel structures. In our study, we determined the expression of Hox genes in postmetamorphic juveniles of T. transversa in order to examine metamorphosis-related changes of expression patterns and to whether Hox genes are expressed in the spatially collinear way in the postmetamor- phic juveniles. Results: Hox genes are expressed in a spatially non-collinear manner in juveniles, generally showing similar patterns as ones observed in competent larvae: genes labial and post1 are expressed in chaetae-related structures, sex combs reduced in the shell-forming , whereas lox5 and lox4 in dorso-posterior epidermis. After metamorphosis, expression of genes proboscipedia, hox3, deformed and antennapedia becomes restricted to, respectively, shell muscu- lature, prospective hinge rudiments and pedicle musculature and epidermis. Conclusions: All developmental stages of T. transversa, including postmetamorphic juveniles, exhibit a spatial non- collinear Hox genes expression with only minor changes observed between pre- and postmetamorphic stages. Our results are concordant with morphological observation that metamorphosis in rhynchonelliformean , despite being rapid, is rather gradual. The most drastic changes in Hox gene expression patterns observed during metamorphosis could be explained by the inversion of the mantle lobe, which relocates some of the more posterior larval structures into the anterior edge of the juveniles. Co-option of Hox genes for the morphogenesis of novel struc- tures is even more pronounced in postmetamorphic brachiopods when compared to larvae. Keywords: Metamorphosis, Hox gene collinearity, Indirect development, Morphology, , , Biphasic life cycle

*Correspondence: [email protected] Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen,

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 2 of 19

Background of the Hox genes, yet there are shifts in the combinations Hox genes encode a family of conserved homeodomain of genes defning particular body regions before and after transcription factors from the ANTP , which by metamorphosis [47, 48]. On the other hand, in other ani- binding to regulatory DNA sequences can activate or mals (especially those with the more pronounced meta- suppress transcription of downstream genes (e.g., [1, 2]). morphosis) only one of the developmental stages exhibits Hox genes are present in genomes of almost all investi- canonical spatial collinearity of Hox genes expression, gated animals (with exception of Porifera, whereas the remaining stage shows either a non-col- and [3–7]) and are hypothesized to represent linear expression or does not express Hox genes at all. a synapomorphy of the consisting of and For instance, in the Hox genes Bilateria [4, 8–10]. In most of bilaterians, Hox genes exhibit spatially collinear expression in the nervous sys- are expressed during embryogenesis, being involved in tem of larvae, whereas in juveniles only the three poste- antero-posterior (A-P) patterning of either the whole rior genes are expressed in the intestine [15]. Conversely or at least some of its developing organ systems in pilidiophoran nemertean Micrura alaskensis and in (e.g., [1, 2, 11]). Interestingly, in the genomes of some indirectly developing enteropneust Schiozcardium cali- animals, the Hox genes are clustered along the chromo- fornicum the specialized larvae develop without express- somes in the same order as they are expressed along A-P ing any of the Hox genes, which, in turn, are expressed axis, a phenomenon referred to as spatial collinearity [2, in the canonical collinear way only in the rudiments of 11–13]. Te clustering of Hox genes in the genome is juvenile worms developing either inside larval body hypothesized as a plesiomorphic feature of Bilateria (e.g., (pilidiophorans) or as the posterior extension of late [13]), which, however, went through extensive remod- (enteropneusts) [49, 50]. A somehow similar situation is eling in some evolutionary lineages (e.g., [12, 14–22]. Yet, found in the indirectly developing Strongylo- spatial collinearity can be preserved despite a disorgani- centrotus purpuratus, in which only two Hox genes (hox7 zation or split of the ancestral Hox cluster (e.g., [14]), the and hox 11/13b) take part in the larva formation, whereas situation for which the term trans-collinearity was coined the rudiments of adult , developing inside the by Duboule [12]. larval body, show collinear expression of fve Hox genes Initially the role of Hox genes has been studied in the (hox7, hox8, hox9/10, hox11/13a and hox11/13b) in the developing embryo of Drosophila melanogaster [23], later extra-axial mesoderm [51, 53–55]. Yet another type of supplemented by the data from other insects, the metamorphosis-related Hox genes expression shifts and [24–26]. Recent advance of molecular is found in scaphopod Antalis entalis in which only the and bioinformatic techniques allowed the investigation of mid- stage exhibits staggered Hox genes Hox gene expression in the embryos and larvae of several expression, whereas both competent larvae and postmet- non-model species, including, e.g., xenacoelomorphs [16, amorphic juveniles lack spatial collinearity [52]. Some of 27, 28], [29], onychophorans [30], tardi- the scaphopod Hox genes partially retain their expression grades [31], [32], [33–35], mollusks [36– profles throughout metamorphosis (hox2, hox5, lox5), 40], nemerteans [41] and brachiopods [19], essentially whereas other substantially changes their expression increasing knowledge on the diversity of Hox gene-based domains (hox3, lox4, post1, post2) or are expressed only patterning systems in Bilateria. before (hox1) or after (hox4) metamorphosis [52]. It is Many animals are characterized by an indirect life therefore evident that the metamorphosis-related shifts cycle in which embryos develop through a larval stage in Hox gene expression and function vary a lot from one and subsequent metamorphosis, during which the larval animal clade to another, as a result of diverse evolution- body is reshaped into the adult one (e.g., [42, 43]). As lar- ary and developmental processes, which shape the ontog- vae and adults can signifcantly difer in their morphol- eny of each particular group [56]. ogy, the transition process might be quite dramatic and One of the animal groups with a distinct metamor- hence attracted attention of many researchers as one of phosis event are rhynchonelliformean brachiopods, the pivotal moments of the animal development [44–46]. represented by T. transversa for which Schiemann et al. Although the process of metamorphosis has puzzled recently described Hox genes expression in embryos numerous developmental biologists, there are relatively and larvae [19]. Brachiopods, along with and few studies regarding shifts of Hox gene expression possibly ectoprocts, constitute the clade Lophophorata accompanying it [15, 47–52]. In some animals, both lar- (Fig. 1A, [57, 58]), which, together with, for example, vae and adults show canonical spatial collinearity, which annelids, mollusks, fatworms, nemerteans and rotifers, often correlates with the gradual type of metamorphosis. belongs to a large clade of animals called Tis can be exemplifed by investigated species, in Spiralia (Fig. 1A, [58–61]). Extant brachiopods are tradi- which both life stages exhibit spatial collinearity of most tionally divided into three groups: , Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 3 of 19

Fig. 1 Phylogenetic position of Brachiopoda (A, based on Laumer et al. [58]), metamorphosis of Terebratalia transversa (B, based on Freeman [68]) and detailed morphology of competent larva (C, based on Santagata [70]). S stands for Spiralia, L for Lophophorata. 1 Competent planktonic larva, anterior to the right; 2 Larva settles on the substrate; 3 Inversion of the mantle lobe in the settled larva; 4 Juvenile; note that over the course of metamorphosis the internal surface of the larval mantle lobe becomes external, shell-covered surface of juvenile animal, external surface of the mantle lobe becomes inner surface of the mantle, whereas anterior lobe contributes to the rudiment developing inside mantle cavity. Musculature in C is depicted in green, nervous system in red and excretory organs in orange. al larval anterior lobe, an anterior nerves, anr anterior nerve ring, cm circular muscle, crs chaetal sac retractor muscle, csm chaetal sac musculature, csn chaetal sac nerve, ds dorsal shell, lgm longitudinal gut-related muscles, lpm lateral pedicle muscle, lr lophophore rudiment, m mantle, ml larval mantle lobe, mo mouth, mpm medial pedicle muscle, np neuropil, nr rudiment, pcn paraxial nerve cord, pe pedicle, pl larval pedicle lobe, pne pedicle nerve, vmm ventral mantle lobe lateral muscle, vs ventral shell Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 4 of 19

Craniiformea and , the two latter form- animals, larvae and juveniles can show collinear expres- ing sister [18, 58, 61, 62], historically united into sion of Hox genes in patterning of one of the life stages, group Inarticulata. As all brachiopods, adults of T. while the other develops without evident Hox expression transversa are flter feeding animals with external anat- collinearity. omy superfcially similar to bivalves—most of the body, Terefore, in this study, we supplemented fndings of including lophophore, a fltering organ, is enclosed in Schiemann et al. [19] by examination of the postmeta- the two-valved shell, which covers the dorsal and ven- morphic Hox gene expression in T. transversa juveniles tral surfaces of the body. Te clade Rhynchonelliformea 2 days after metamorphosis. Te main questions, which is further characterized by the set of morphological fea- we were aiming to answer, were: (1) If and how is Hox tures, including posterior soft-tissued pedicle (by which genes expression pattern shifted during metamorpho- animal attaches to the substrate), blind gut devoid of sis in rhynchonelliformean brachiopods? (2) Is there and articulated valve-hinge [63]. Additionally, rhyn- any staggered Hox genes expression along the A-P axis chonelliformean larvae (Fig. 1B1, C) difer from those emerging after metamorphosis as a result of displace- found in other brachiopods by possessing three dis- ment of larval Anlagen and their development into def- tinct body regions—anterior lobe, mantle lobe (bearing nite adult structures? four chaetal sacs) and the most posterior pedicle lobe [63–67]. Te rhynchonelliformean larva settles by adher- ing to the substrate with the posterior tip of the pedicle Results lobe (Fig. 1B2) and undergoes a specifc metamorpho- Description of T. transversa juvenile morphology sis, which in case of T. transversa is relatively rapid (few Existing knowledge of the detailed morphology of the hours to 1 day [64]) and involves inversion of the man- juvenile T. transversa is based mostly on the confocal tle lobe (Fig. 1B2–3, [64, 68]). Te latter results in pro- laser scanning microscopy (CLSM) investigation of mus- found relocation of some larval tissues—in competent culature [70, 71], as well as transmission electron micro- larvae the mantle lobe partially covers the pedicle one scope (TEM) sections [64, 69, 72] and scanning electron and its chaetae projects posteriorly, after metamorphosis microscopy (SEM) [69, 72, 73] of diferent developmen- mantle lobe with chaetae projects anteriorly, its former tal stages, including juveniles 1 day after metamorphosis interior surface becomes exposed and produce protegu- [69, 70, 73], 4–5 days after metamorphosis [69, 71–73] lum (the frst rudiment of the shell), whereas its former and older than 1 week after metamorphosis [69, 70, 72, exterior surface constitutes walls of the mantle cavity of 73]. Terefore, to facilitate interpretation of our gene the juvenile (Fig. 1B2–4) [64, 68, 69]. Terefore, the rapid expression results [74], we examined morphology of transition from larvae to the juvenile involves profound the juveniles 2 days after metamorphosis utilizing light reshaping of the entire body, which poses a question to microscopy (LM) and CLSM combined with DAPI, phal- which extent are those two stages continuous [70]. loidin and immunohistochemical stainings (with primary Schiemann et al. investigated genomic order of Hox antibodies against tyrosinated and acetylated tubulin). genes of T. transversa and Hox genes expression in Two days after metamorphosis, the juveniles of T. embryos and larvae of T. transversa and craniiformean transversa already resemble the adult animal in their gen- anomala [19]. T. transversa has a split Hox eral shape (Fig. 2A–C). Te body is clearly divided into cluster comprising of 10 Hox genes in three independ- main part covered by the two-valved juvenile shell and a ent parts. One scafold contains two anterior Hox genes, posterior pedicle (pe, Fig. 2A–C), by which the juvenile is labial (lab) and proboscipedia (pb). A separate scafold attached to the substrate. contains the longest section of the Hox complex, con- Anteriorly, the shell is lined with the mantle margin taining genes hox3, deformed (dfd), sex combs reduced (mr, Fig. 2B, C), where the tissues responsible for the (scr), lox5, antennapedia (antp), lox4 and post2, whereas of the prospective adult shell are localized [69]. the most posterior gene post1 is located in the third Phalloidin staining revealed the presence of the devel- independent scafold [19]. A disorganization of the Hox oping mantle margin muscles (arrowheads Fig. 2D; mm, cluster has also been reported for linguliformean brachi- Figs. 2G, 3C, C’), which have been already described for opod, anatina, in which although all Hox genes the older juveniles [70, 71]. Additionally, four chaetal sacs are in the single cluster post1, post2, lox4 and antp have (cs, Fig. 2A–C), associated with the degenerating larval been translocated upstream to the lab [18]. In embryos musculature (csm, Figs. 2D, G; 3D, D’) [70], are embed- and larvae of T. transversa, but also of craniiformean N. ded in the dorsal mantle margin, one pair dorso-medially anomala, detected expression pattern of Hox genes does and another in the more lateral position, which, respec- not show the canonical spatial collinearity [19]. How- tively, protrude numerous chaetae (ch, Fig. 2A, C) anteri- ever, as stated before, in some indirectly developing orly and laterally. Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 5 of 19

(See fgure on next page.) Fig. 2 Morphology of the juvenile of Terebratalia transversa (2 days after metamorphosis), visualized with light microscopy (A) and CLSM (B, D–I). A Micrograph of the entire animal. B Frontal section through the median part of animal, cell nuclei visualized with DAPI staining. C Schematic drawing of the of juvenile in dorso-ventral (top) and lateral (bottom) views, musculature in green, tyrosinated-tubulin immunoreactive nervous system in red, acetylated-tubulin immunoreactive structures in orange (mantle margin ciliation not shown for clarity). D, G Musculature visualized with F-actin phalloidin staining, arrowheads in D point to the ventral mantle margin muscles. E, J Tyrosinated-tubulin immunoreactivity. F, I acetylated-tubulin immunoreactivity. H Transverse section through the nephrostome, cell nuclei visualized with DAPI in cyan, acetylated-tubulin immunoreactivity in yellow. A–F Dorso-ventral view, anterior to the top. G Lateral view, anterior to the left, dorsal to the top. I Virtual transverse section through the middle part of animal, dorsal to the top. J Ventral, magnifed view of the commissural region from E. aam anterior shell adductor muscle, asc anterior supraesophageal commissure, ch chaeta, cr rudiment, cs chaetal sac, csm chaetal sac musculature, dc dorsal commissure, dms shell diductor muscle, dt digestive tract, lr lophophore rudiment, lrf fbers in the lophophore rudiment, mc mantle cavity, mm mantle margin musculature, mmc mantle margin ciliation, mr mantle margin, nd nephroduct, np neuropil, ns nephrostome, pam posterior shell adductor muscle, pe pedicle, psc posterior supraesophageal commissure, vmc ventral mantle cavity ciliation, vpa ventral pedicle adjustor muscle

Optical sections through the animal show the narrow Two prominent tTIR and aTIR longitudinal structures mantle cavity (mc, Figs. 2B, C, 3C’–G’), which expands are present in the ventro-lateral part of the animal (nd, ventro-medially to about two-thirds of the length of the Fig. 2C, F, I), extending along the ventral surface from animal body and is lined with the ciliated cells (vmc, the mid-posterior region to the ventro-posterior part of Fig. 2C, F, I). Te remnant of the larval anterior lobe, the mantle cavity. Dorsally those structures have numer- from which the prospective lophophore will develop [66, ous fnger-like projections (ns, Figs. 2H, I, 3G, G’), which 75], is situated inside the mantle cavity (lr, Figs. 2B, C, 3B, contact nuclei-free regions (as revealed by DAPI stain- B’, C, C’). Posteriorly the lobe is connected with the dorsal ing, Fig. 2H). We suggest that those structures represent mantle, and ventrally it faces the extension of the mantle metanephridia (composed of nephrostome and nephrid- cavity (Fig. 3D, D’, E, E’). Medially the lobe is divided by ial duct) of the juveniles, which connect the developing the ciliated slit (cs, Fig. 2F, I), which anteriorly commu- coelom with the mantle cavity. Teir form and position nicates with the mantle cavity through the ventral infold, are similar to what has been described for the metane- a stomodeum (Fig. 3 C–E, C’–E’) and posteriorly con- phridia of relatively closely related Terebratulina retusa tinues as the tubular rudiment of the digestive tract (dt, [76]. Although metanephridia in brachiopods are con- Figs. 2A–C, 3F, F’). sidered to be responsible only for release of and At this stage, the lophophore rudiment is poorly devel- not for excretion [76], they are present (albeit initially as oped and consists of two, scarcely ciliated lobes without non-functional rudiments) already in the early juveniles tentacles (lr, Figs. 2B, C, 3B, B’, C, C’). Te lobes are pen- of N. anomala [77]. It is possible that aTIR structures etrated by numerous, fne tyrosinated-tubulin immuno- described by Santagata [70] as larval protonephridia in reactive (tTIR) fbers (lrf, Fig. 2C, E), which communicate T. transversa (nr, Fig. 1C) actually represent rudiments of with the nervous system and probably represent the the metanephridial ducts or nephrostomes which acquire developing innervation of the prospective lophophore. their fnal form during or soon after metamorphosis. Te most prominent structure of the nervous system Te DAPI staining revealed an empty cavity inside the is the brain neuropile (np. Fig. 2C), which consists of body of the juvenile with two pairs of anterior and pos- two tTIR and acetylated-tubulin immunoreactive (aTIR) terior branches (cr, Figs. 2B, C, 3), which most probably commissures (anterior and posterior supraesophageal represents the developing coelom in which some of the commissures, respectively, asc and psc, Figs. 2C, E, I, J, forming muscles are freely positioned (Fig. 3E, E’, H, H’) 3E, E’, F, F’), positioned dorsally to the ciliated slit. Some [78]. Its two anterior branches extend along digestive tTIR and aTIR fne fbers extend laterally from those tract and penetrate the lophophore rudiment (Figs. 2B, commissures to the lophophore rudiments (lrf, Fig. 2C, 3C–F, C’–F’). A similar arrangement of the coelom in the E) and mantle tissues (including mantle margin and lophophore rudiment of postmetamorphic juveniles has chaetal sacs, Fig. 2C). Few tTIR fne neurites extend from been described for relatively closely related rhynchonel- the posterior supraesophageal commissure to the intes- liformean Calloria inconspicua [79]. tinal tissue (arrowheads, Figs. 2J, 3F’). Additional dorsal In addition to the already mentioned musculature tTIR commissure (dc, Figs. 2C, E, J, 3E, E’) connects with related to the mantle margin, we identifed rudiments of the anterior supraesophageal commissure. Te similar all the muscle groups (pedicle adjustors, shell diductors arrangement of the nervous system in the early juve- as well as anterior and posterior shell adductors, respec- niles of T. transversa has been reported based on immu- tively, vpa, dms, aam, pam, Figs. 2D, G, 3D–H, D’–H’) nostaining against serotonin [28]. described for the older juveniles of T. transversa [71] with Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 6 of 19 Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 7 of 19

(See fgure on next page.) Fig. 3 Transverse sections through Terebratalia transversa juvenile (2 days after metamorphosis), showing detailed morphology of the animal. Each panel consists of CLSM image (left) and schematic representation (right). Cell nuclei visualized with DAPI in cyan, acetylated-tubulin immunoreactivity (F and G) and tyrosinated-tubulin immunoreactivity (E) in yellow, F-actin phalloidin staining in green. A Juvenile in dorso-ventral view, anterior to the top, dashed red lines indicates sections shown on the subsequent panels. B–H Virtual transverse sections through the juvenile, as indicated on A, dorsal to the top on all panels, on the right panels outline of the body is depicted in light blue, musculature in green, body cavities in gray, nervous system in red and excretory organs in orange. Scale bars on all images represent 20 μm. aam anterior shell adductor muscle, asc anterior supraesophageal commissure, cr coelom rudiment, csm chaetal sac muscle, dc dorsal commissure, dm dorsal mantle, dms shell diductor muscle, dt digestive tract, lr lophophore rudiment, mc mantle cavity, mm mantle margin muscle, np nephropore, ns nephrostome, pam posterior shell adductor muscle, psc posterior supraesophageal commissure, vm ventral mantle, vpa ventral pedicle adjustor muscle

the only exception of the lophophore-related tentacle (Fig. 5F–H) indicates that dfd is expressed in the ventral muscles (which correlates with lack of the lophophore pedicle adjustor muscles (compare Figs. 2C, D, G, 5F–H). tentacles 2 days after metamorphosis). In diferent speci- Expression of the gene scr is restricted to the mantle mens, the particular groups of muscles were developed margin (Figs. 4E, 5I). Signal from probes against scr in to diferent degree corroborating the observation of the CISH seems to be diversifed into smaller domains with extensive and rapid remodeling of muscular tissue in the strong signal interspaced by wider regions of relatively postmetamorphic juveniles [70]. weaker expression (respectively, arrowheads and aster- isks, Fig. 4E), indicating an unequal expression of the In situ hybridization of Hox genes gene along mantle margin. However, this diversifcation Te expression of the Hox genes in juvenile T. transversa is not visible in examination (compare Figs. 4E, 5I). (2 days after metamorphosis) was examined with colori- Te uniform signal from probes against scr in FISH might metric (CISH; Fig. 4) and fuorescent (FISH, Fig. 5) in situ be an efect of the specifc staining of the scr expressing hybridization. Hox genes in the juveniles of T. transversa cells and unspecifc binding of the probe at the mantle are not expressed in a strictly collinear way (Figs. 4, 5, 6). margin (as in fuorescent stainings against lab and pb, Te most anterior Hox gene lab is expressed in the where unspecifc signal is visible along mantle margin; two bilaterally paired domains at the mantle margin, compare Fig. 5I with 5A, C). which correspond to the larval chaetal sacs (arrowheads, Te gene lox5 is expressed in the continuous dorso- Figs. 4A, 5A, B; also compare with Fig. 2B, D). posterior domain, which extend from posterior region of Te gene pb has bilaterally paired strong expression the shell-covered body (asterisks, Figs. 4F, 5J) to the pedi- domains (arrowheads, Figs. 4B, 5C), which correspond to cle tissues (arrowheads, Figs. 4F, 5J) and its expression the position in which shell adductor muscles are devel- is restricted to the dorsal epidermal cells as revealed by oping (compare Figs. 2D, 4B, 5C). Te FISH and CLSM FISH (double arrowheads, Fig. 5K). investigation of juveniles further revealed that those two antp has a distinct expression domain only in the epi- domains extend obliquely from the more anterior point dermis of the pedicle, as revealed by both CISH (arrow- on the dorsal shell to the more posterior point on the head, Fig. 4G) and FISH (arrowhead, Fig. 5N–P). Te ventral shell, in the same orientation as the anterior shell signal in the CISH staining developed for the long time adductors (compare Figs. 2C, G, 5D). and before it became evident the strong staining had hox3 is expressed in two paired domains posteriorly appeared in some specimens also in the dorso-posterior to the most lateral projections of the shell (arrowheads, part of the shell-covered body. However, the control with Figs. 4C, 5E), where prospective hinge rudiments will sense probe showed that this staining results from unspe- form in the older juveniles [69]. CISH investigation cifc binding of the probe in the dorsal protegulum (larval showed additional broad weak staining in the posterior shell rudiment; asterisks, Additional fle 1: Figure S1B) part of the body (Fig. 4C), which was not reproduced and on the borders between the dorsal protegulum and with FISH (Fig. 5E) and which might result from unspe- the remaining parts of the shell (arrowheads, Additional cifc probe binding in the posterior shell as shown by fle 1: Figure S1B). Te strong dorsal band was also vis- sense probe staining (Additional fle 1: Fig. S1B and C). ible in FISH staining (double arrowheads, Fig. 5N), but Te gene dfd is expressed in the ventro-posterior combined staining with DAPI showed that it is restricted domain (Figs. 4D, 5F–H) composed of extensive lateral to the surface area and does not penetrate the epidermis elements (arrowheads, Figs. 4D, 5F–H), which merge (arrowhead, Additional fle 1: Fig. S1D, E), supporting our posteriorly (connection visible only with CISH, asterisks fnding that it represents an unspecifc probe binding by Fig. 4D). Position of those structures revealed with FISH shell components. Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 8 of 19 Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 9 of 19

Fig. 4 Whole-mount colorimetric in situ hybridization of the Hox genes (A–J) and schematic representation of expression patterns (K) in Terebratalia transversa postmetamorphic juveniles (2 days after metamorphosis). All micrographs and drawings in dorso-ventral view, anterior to the top. For each plate (A–J) name of the hybridized gene is provided in the lower right corner. Particular structures in which each of the genes is expressed are indicated with arrowheads, asterisks and double arrowheads (see text for detailed explanation). Note that signal on I (asterisk) represents unspecifc background (see text for details) Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 10 of 19

(See fgure on next page.) Fig. 5 Whole-mount fuorescent in situ hybridization of the Hox genes (red) combined with DAPI staining of cell nuclei (cyan, with exception of E and N) in Terebratalia transversa postmetamorphic juveniles (2 days after metamorphosis). For each plate name of the hybridized gene is provided in the right corner. Particular structures in which each of the genes is expressed are indicated with arrowheads, asterisks and double arrowheads (see text for detailed explanation). Expression of lab in chaetal sacs in dorso-ventral view (A) and virtual cross section (B); note autofuorescence of chaetae. Expression of pb in anterior shell adductor muscles in dorso-ventral view (C) and virtual parasagittal section (D). Expression of hox3 in dorso-ventral view (E). Expression of dfd in ventral pedicle adjustor muscles in dorso-ventral view (F), virtual cross section (G) and parasagittal section (H). Expression of scr in mantle margin (I). Expression of lox5 in dorso-posterior epidermis in dorso-ventral view (J) and virtual sagittal section (K). Expression of lox4 in dorso-posterior epidermis in dorso-ventral view (L) and virtual cross section (M). Expression of antp in pedicle tissue in dorso-ventral view (N, O). Expression of post2 in mantle margin, dorso-ventral view (P). Dashed lines with letters on A, C, F, J, L and O indicate section planes shown on respective plates. Scale bars on all images represent 20 μm. Anterior to the top on A, C, E, F, I, J, L, N and R; dorsal to the top on B, D, G, H, K and M; anterior to the left on D, H and K.

Te central-class Hox gene lox4 is expressed only in Brachiopoda [19], whereas arx is expressed in the chaetal the small area of epidermal tissues in the dorso-posterior sac musculature of annelid Platynereis dumerilli [81] part of the shell-covered body (arrowheads, Figs. 4H, 5L, and in the developing chaetal sacs of T. transversa [19]. M), and its expression domain is not extending to the We did a double fuorescent staining of post1 and labial pedicle tissues. (Fig. 6A) as well as post1 and arx (Fig. 6B) in the late We did not manage to detect expression of post2 gastrula stage and lab and arx in the early trilobed larva with in situ hybridization, which corresponds to the (Fig. 6C). reported overall low level of post2 transcription in post- In the late gastrulae, gene post1 is co-localized with metamorphic juveniles of T. transversa [19]. After long lab (Fig. 6A3), which shows extremely weak expression developmental time, CISH staining yielded signal in the at this developmental stage (asterisks, Fig. 6A2). Tis is dorso-posterior part of the shell-covered body (aster- concordant with the CISH results from Schiemann et al. isk, Fig. 4I); however, the control with the sense probe [19]), but our results show that it is also expressed in showed that this results from unspecifc binding of the some post1-negative cells in between chaetal sac Anla- probe in the larval dorsal protegulum (Additional fle 1: gen (asterisks, Fig. 6A3). Additionally post1-positive cells Figure S1C). Te FISH staining only revealed a signal at of the late gastrulae strongly express gene arx (Fig. 6B). the borders of the larval protegulum and the remaining In the early trilobed larvae lab is expressed in the chaetal parts of the shell (arrows, Additional fle 1: Fig. S1F) and, sac-related cells (Fig. 6C1), whereas arx expression is similarly as in case of antp, FISH combined with DAPI restricted to the subpopulation of the cells of the inner staining revealed that this signal is restricted to the sur- mantle lobe epithelium (Fig. 6C2) and the two genes are face (shell components) and does not penetrate to the not co-expressed by any cells (Fig. 6C3). cellular epidermal layer (arrowhead, Additional fle 1: Figure S1G). Te unspecifc binding of some probes by Discussion the larval protegulum has been already reported for T. Metamorphosis and Hox gene expression transversa larvae [80], and apparently this phenomenon in Rhynchonelliformea can also pose a problem in investigation of postmetamor- Comparison of the expression of Hox genes between phic animals. late, competent larva and postmetamorphic juvenile of Expression of the most posterior Hox gene post1 is T. transversa (Fig. 7) shows that in both stages almost all detected along mantle margin (arrowheads, Figs. 4J, 5R), Hox genes (with the exception of hox3, post2 and post1) showing a relatively equal strength of signal with both are expressed in the corresponding organs and body CISH and FISH. regions: lab in chaetal sacs, pb and dfd in mesoderm, scr in the shell growth zone, whereas lox5, antp and lox4 Double fuorescent in situ hybridization are expressed in the dorso-posterior ectoderm. Most of the chaetae‑related genes of the observed diferences and shifts in the expression In addition to the investigation of Hox genes in postmeta- domains can be explained by the inversion of the mantle morphic juveniles, we performed double FISH of genes lobe, which constitutes the most profound process dur- lab, post1 and arx (Aristaless-related homeobox) at the ing the whole metamorphosis in Rhynchonelliformea early developmental stages of T. transversa in order to (Fig. 1B, [64]). Another factor, which contributes to the better understand the relation of the expression patterns observed changes, is the restriction of the expression of to the chaetal sac formation. Te two former Hox genes some Hox genes from broad, less specifc larval domains have been proposed as related to chaetae formation in to the particular structures of the juvenile, which emerge Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 11 of 19 Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 12 of 19

Fig. 6 Expression of the chaetae-related genes in the early developmental stages of Terebratalia transversa combined with DAPI staining of cell nuclei (cyan). Double fuorescent in situ hybridization of post1 and lab (A), post1 and arx (B) and lab and arx (C) in the early gastrulae (A, B) and the early trilobed larva (C). Dorso-ventral view and anterior to the top on all panels. Scale bars on all images represent 20 μm

during or after metamorphosis. For example, pb is gen- Te comparison of the Hox gene expression between erally expressed in the anterior mesoderm in late larvae larvae and juveniles allows the identifcation of the Anla- but in juveniles its expression becomes restricted only gen of adult structures in the larva. For example, the to particular mesodermal structures, i.e., newly formed expression patterns of the Hox genes before and after anterior shell adductors muscles. metamorphosis suggest that only the posterior part of Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 13 of 19

Fig. 7 Comparison of the Hox genes expression between the late competent larva (A, based on Schiemann et al. [19]) and juvenile (B) of Terebratalia transversa. Animals are shown in the dorso-ventral view (right panels) and in the lateral view with dorsal to the right (left panels). Anterior to the top on all panels. Bars on the right show antero-posterior Hox gene expression gradients in ectoderm and mesoderm of each developmental stage the larval pedicle lobe contributes to the pedicle of the undergoes more or less pronounced metamorphosis [63, adult, whereas the more anterior part becomes the pos- 70, 83–87]. In Linguliformea, the metamorphosis itself is terior region of the shell-covered body, as it has been extended over time with some of the juvenile traits pre- proposed by Stricker and Reed [64, 72]. Among six Hox sent already in the planktotrophic larvae [67, 70, 83, 88], genes expressed in the pedicle lobe of the late larvae of and the most advanced larval stages are even commonly T. transversa, only lox5 and antp are expressed in the considered as representing planktonic juveniles or par- pedicle of the postmetamorphic juvenile (Fig. 7), both of alarvae [63, 70, 88]. One can therefore speculate that as them being expressed in the most posterior part of the larval and adult body plans in Linguliformea are continu- larval pedicle lobe [19]. ous, their patterning by Hox genes should be similar as is Next to Rhynchonelliformea, two inarticulate clades a case in T. transversa. On the other hand, there are two belong to Brachiopoda: Craniiformea and Linguli- competing hypotheses about nature of the rearrange- formea [82], both possessing a planktonic larvae, which ment of the larval body plan during metamorphosis of Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 14 of 19

craniiformean brachiopods [63, 84, 85, 89, 90]. Te main Brachiopoda [62, 82]) and their rapid metamorphosis controversy regards whether the N. anomala larva, which involves drastic rearrangements of the larval body plan lacks the distinct pedicle lobe, attaches to the substrate [67, 94–97], which is much more complicated than the with its dorso-posterior side [84] or with the posterior transition found in Rhynchonelliformea and sometimes tip of the posterior lobe [85]. Expression of the Hox genes referred to as catastrophic or cataclysmic metamorphosis is relatively similar between embryos and larvae of N. [94, 96, 98]. Te recent analysis of the body region-spe- anomala and corresponding stages of T. transversa [19], cifc transcriptomes revealed that in adults of Phoronis indicating a conserved nature of Hox genes patterning austarlis, which possesses an organized Hox cluster, between Craniiformea and Rhynchonelliformea. lox5 and Hox gene expression does not exhibit spatial collinearity antp, which after metamorphosis are expressed in the [18]. Unfortunately, data on the spatial expression of Hox pedicle of T. transversa juveniles are expressed in the pos- genes in early developmental stages of any spe- terior tip of the posterior lobe of N. anomala larvae [19] cies are still lacking [98], preventing analysis of metamor- favoring interpretation that posterior tip of N. anomala phosis-related Hox genes expression shifts. Nevertheless, larvae corresponds to the pedicle of Rhynchonelliformea it is possible that in phoronids the larvae and juveniles [85, 90]. Further investigation of the postmetamorphic exhibit pronounced diferences in the Hox genes expres- expression of Hox genes, especially lox4 and antp, in N. sion as is a case in some other animals with catastrophic anomala could support this hypothesis. and extensive metamorphosis [15, 49, 50]. Unlike some bilaterians in which metamorphosis seems to be related to highly diferent Hox gene expres- Germ layer‑specifc expression of Hox genes sion between larvae and adults (e.g., [15], Bry- In most of the investigated Bilateria, Hox genes are pre- ozoa [91], scaphopods [52]) or in which Hox genes are dominantly expressed in the ectodermal domains and not expressed in the larvae and only pattern adult body often their antero-posterior staggered expression is espe- (pilidiophoran nemerteans [49], indirectly develop- cially evident in the neuroectoderm, which lead to the ing Hemichordates [50], sea urchins [51, 53, 54]), rhyn- assumption that at least one of the original roles of Hox chonelliformean brachiopods exhibit continuity in the genes was patterning of the developing nervous system patterning of larval and adult body plans. Consequently, along A-P axis [27, 32, 99]. Interestingly, we did not fnd in regard to Hox gene expression, metamorphosis in T. any of the Hox genes expressed in the nervous system of transversa is similar to the condition found in another postmetamorphic juveniles of T. transversa. Tis could spiralian clade, Annelida. Although there are some shifts be explained by the fact that in juveniles the main nerv- in expression patterns of particular Hox genes between ous structures are brain and lophophore nerves (Fig. 2C) annelid larvae and juvenile worms [47, 48], those dif- both related to the larval anterior lobe and postmeta- ferences are mostly related to restriction of some of the morphic lophophore rudiment, which represent deriva- genes from broader larval to more specifc adult domains tives of the head and hence do not express Hox genes (the [47]. Tis similarity can be explained if one assumes that, same has been shown for the phoronid lophophor [18]). same as in Annelida, the metamorphosis of rhynchonel- Schiemann et al. [19] also did not describe expression of liformean larvae is not as drastic as it might seem and any of the Hox genes in neuroectoderm of earlier devel- instead represents a relatively gradual process [67]. In T. opmental stages of T. transversa. However, as co-expres- transversa, several of the adult structures, including shell sion of neuroectoderm markers has not been tested in secreting epithelium [64, 68] or pedicle muscles [70, 72], that work, it is difcult to ascertain whether T. transversa are already present in the competent larvae as the Anla- really lack Hox genes expression in neuroectoderm on all gen. Tus, even though transition from larva to juvenile developmental stages. poses large ecological change, from the morphological Hox genes can be also expressed in particular mesoder- point of view the mantle lobe inversion is related mostly mal domains in almost all investigated bilaterians, with to tissue relocation and not to the degeneration or forma- the exception of Hemichordates (where their expression tion of entire body regions, as is the case in pilidiophoran is restricted to ecto- and endoderm [29, 50]), rotifers nemerteans, indirectly developing hemichordates and sea (expression exclusively in the nervous system [32]) and urchins or ascidians. Nemerteans (expression in ecto- and neuroectoderm From the phylogenetic and developmental point [41]). Whether Hox genes were ancestrally expressed of view, it would be interesting to compare shifts of in the bilaterian mesoderm remains an open question. Hox genes expression observed during metamorpho- Nevertheless, taking into account that set of Hox genes sis between T. transversa and Phoronida. Phoronids expressed in the mesodermal derivatives difers substan- are closely related to brachiopods [57, 58, 92, 93] (in tially from one animal group to another and that their past even proposed as specialized clade belonging to transcription in mesodermal tissues can happen on very Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 15 of 19

diferent developmental stages, it seems plausible that study, we detect expression of lab in the chaetal sacs of Hox genes have been recruited many times indepen- juveniles as well, but surprisingly we found that post1 is dently to act in mesoderm development and specifcation also expressed in the postmetamorphic juveniles. Moreo- [99]. ver, its expression is not only restricted to the chaetal sacs In Brachiopods, three of the Hox genes (pb, hox3 and but instead could be detected in the entire marginal zone dfd) show mesodermal expression albeit all of them are of the mantle. Tis fnding, however, makes sense when also expressed in ectodermal domains at some point of one takes into consideration that as adults T. transversa, development [19]. Prior to the metamorphosis, hox3 as most of the rhynchonelliformean brachiopods, possess and dfd are expressed both in the mesodermal and ecto- numerous chaetae along the mantle margin [88, 104]. dermal structures and after metamorphosis, due to the We therefore propose that although both lab and post1 restriction of broader domains into specifc structures, are involved in the chaetae formation in Rhynchonel- hox3 remained expressed only in the ectoderm, whereas liformea, they play diferent roles: post1 is expressed dfd become restricted to the mesoderm (Fig. 7). in the regions where prospective chaetae will develop, Orthologs of those three genes are reported as meso- possibly stimulating epidermal cells to diferentiate into dermally expressed in some other spiralian species as chaetal sacs before its expression decays. A similar role well. For instance, pb exhibits mesodermal expression has been suggested for post1 in annelids, whose chaetae domains in gastropod Haliotis asinina [36] and two are considered homologous to brachiopod ones based annelids—Chaetopterus variopedatus [33] and Alitta on morphological [105] and molecular [19] similari- virens [48], hox3 and dfd are expressed in the mesoderm ties. In annelids, post1 is expressed in the cells of devel- of scaphopod Antalis entalis [52] and hox3 is mesoder- oping chaetae-bearing parapodia, but the expression mally expressed in annelid [47]. However, becomes more faint over the time of development and is lack of evidence that all three of those three genes are not detectable in the already formed parapodia [34, 35, expressed in the mesoderm of any single non-brachiopod 47]. lab, on the other hand, is possibly involved in the spiralian species as well as diferent timing of their meso- patterning of the growth of the chaetae itself, remaining dermal expression in particular animals indicates that expressed long after onset of chaetal sac formation. Tis expression of pb, hox3 and dfd in developing mesoderm hypothesis needs to be tested in the future by functional might represent apomorphic feature of Brachiopoda or gene inference and the examination of older juveniles or Lophophorata (investigation of phoronids and ectoprocts adults, in which, accordingly, we would expect lack of is needed to ascertain). post1 expression and broad expression of lab along the entire mantle margin. Expression of Hox genes during the morphogenesis Additionally, our investigation of the expression of of brachiopod‑specifc structures in T. transversa chaetae-related genes in the earlier developmental stages Although Hox genes are believed to originally be respon- of T. transversa shows that process of chaetal sacs for- sible for antero-posterior patterning [1, 2, 11], in certain mation is complicated and involves cell types, which animal lineages some of them were co-opted for mor- spatially and temporarily difer in their gene expression phogenesis of evolutionary novel structures [100–103]. profles. At the late gastrula stage, lab, post1 and arx are Among Spiralia, such phenomenon has been reported in, all expressed in the two pairs of cell clusters, which have e.g., conchiferan molluscs [36–38, 52] and annelids [34, been interpreted as chaetal sacs Anlagen by Schiemann 35, 47, 48], whereas recently Schiemann et al. suggested et al. [19]. In the later larval stage, only expression of lab that in brachiopod larvae 4 out of 10 Hox genes have is retained in the chaetal sacs-related cells, post1 is not been recruited for patterning of chaetae (lab and post1) expressed anymore, whereas expression of arx is shifted and shell felds (scr and antp) [19]. Our results generally to the inner mantle lobe epithelium, which secrets pro- support fndings of Schiemann et al.—although we did tegulum (the larval shell rudiment). Interestingly, arx is not fnd evidence for the expression of antp in the shell not only expressed in the chaetal sacs Anlagen of anne- feld—and show that co-option of Hox genes for morpho- lids [81] and brachiopods and in the protegulum secret- genesis of novel structures is even more pronounced in ing epithelium of brachiopods but also in the radula juveniles of T. transversa than it is in the larvae. formative tissue of the gastropod Tylomelania sarasino- lab and post1 are recruited for the morphogenesis of rum [106]. Tis indicates that among lophotrochozoans chaetae in the embryos and larvae of T. transversa [19]. arx is generally expressed in the tissues forming various Te gene lab is constantly expressed in the chaetal sacs hard structures and cannot be unambiguously related to from formation of their early Anlagen up to the latest lar- only single type of them. val stage, whereas post1 is only briefy expressed during Te two-valved shell and posterior pedicle represent short time window, when the Anlagen are formed. In our two distinct apomorphies of brachiopods, and we found Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 16 of 19

four out of ten Hox genes expressed in the structures larvae were cultured following previously published pro- related to those morphological novelties. Our results tocols (e.g., [19, 64, 68]) up to the metamorphosis. Two indicate that scr is likely co-opted for the juvenile shell days after metamorphosis, juvenile animals were gently formation, as the gene is expressed in the mantle mar- scraped from the bottom of the dish with a razor blade, gin in the region specialized for shell secretion [69]. Tis relaxed with ­MgCl2, fxed in 3.7% formaldehyde and fnding corresponds to the results of Schiemann et al. washed in phosphate bufer. Fixed animals were stored in [19], who found expression of scr in the epithelial cells 100% methanol. forming larval shell rudiment. Both shell and pedicle require sets of specialized mus- In situ hybridization cles, which constitute an important part of the brachio- Probes against Hox genes were synthesized using the pod body. In the late larvae of T. transversa, the genes pb same plasmid clones as used in Schiemann et al. [19], and dfd are likely responsible for A-P patterning of meso- where the gene orthology assessment has been per- derm [19], yet during postembryonic development they formed. Single whole-mount in situ hybridization was seem to be recruited into morphogenesis of specifc mus- performed following an established protocol [107]. cular structures that drive the biomechanics of, respec- dUTP-digoxigenin-labeled probes were hybridized at a tively, shell and pedicle. Additionally, hox3, another gene concentration of 1 ng/μl at 67 °C for 72 h, detected with that seems to play a role in mesoderm patterning dur- anti-digoxigenin-AP antibody in 1:5000 concentration ing earlier developmental stages [19], is expressed in the in blocking bufer and visualized with nitroblue tetrazo- regions where future hinge rudiments will develop [69], lium chloride and 5-bromo-4-chloro-3-indolyl phosphate suggesting that it could be involved in the morphogenesis (in colorimetric in situ hybridization) or detected with of this autapomorphic rhynchonelliformean feature. anti-digoxigenin-POD antibody in 1:200 concentration in blocking bufer and visualized with TSA-Cy5-Plus (in Conclusions fuorescent in situ hybridization). Additionally, animals All developmental stages of T. transversa, including prepared for FISH were stained for 30 min in DAPI to juveniles, express Hox genes in a spatially non-collinear visualize cell nuclei. Stained juveniles where mounted manner [19]. Most of the patterns observed in the late in 70% glycerol and examined with Zeiss Axiocam HRc larvae seem to persist throughout metamorphosis and connected to a Zeiss Axioscope Ax10 using bright-feld are retained in juveniles, corroborating morphological Nomarski optics (CISH) or scanned in Leica SP5 confo- observations that metamorphosis, despite being rapid, is cal laser scanning microscope (FISH). Double fuores- of gradual type and most of the adult organs are present cence in situ hybridization was conducted as described as Anlagen in the competent larvae. Te most drastic elsewhere [108]. shifts in Hox gene expression patterns observed during metamorphosis can be explained by: (1) the inversion of Immunohistochemistry the mantle lobe which relocates some of the more poste- For investigation of juvenile morphology, mouse pri- rior larval structures into the anterior edge of the juve- mary monoclonal antibodies against tyrosinated-tubulin niles and (2) restriction of the broad expression domains, (Sigma, T9028) and acetylated-tubulin (Sigma, T6793) present in larvae, to the specifc structures in juveniles. were used in 1:500 concentration. To visualize the pri- Concordantly to the previous study on larvae of T. mary antibodies, secondary goat anti-mouse antibodies transversa, we found that certain Hox genes have been (Life Technologies) conjugated with fuorochrome (Alex- evolutionary co-opted for morphogenesis of special- aFluor647) were applied in 1:50 concentration. F-actin ized structures in brachiopods. In both larvae and juve- was visualized with AlexaFluor555-labeled phalloidin, niles, lab is expressed in the chaetal sacs, whereas post1 and cell nuclei were stained with DAPI. Stained juveniles marks the area where prospective chaetae will develop. In were mounted in 80% glycerol and scanned in Leica SP5 juveniles, four out of the ten Hox genes are expressed in confocal laser scanning microscope. the epidermal (scr, hox3) and muscular (pb, dfd) tissues related to shell and pedicle, two autapomorphic features Image processing and fgure preparation of Brachiopoda. Z-stacks of confocal scans were projected into 2D images and 3D reconstructions in IMARIS 9.1.2. Both light Methods micrographs and CLSM images were adjusted in Adobe Animal collection and fxation Photoshop CS6 and assembled in Adobe Illustrator CS6. Gravid adults of T. transversa (Sowerby 1846) were col- All the schematic drawings were done with Adobe Illus- lected near San Juan Island, Washington, USA. Eggs trator CS6. obtained from the animals were fertilized, and developing Gąsiorowski and Hejnol EvoDevo (2019) 10:1 Page 17 of 19

Additional fle of the ctenophore Mnemiopsis leidyi suggests that Ctenophora and Porifera diverged prior to the ParaHoxozoa. Evodevo. 2010;1(1):9. 5. Jakob W, Sagasser S, Dellaporta S, Holland P, Kuhn K, Schierwater Additional fle 1: Fig. S1. Background signal resulting from unspecifc B. The Trox-2 Hox/ParaHox gene of (Placozoa) marks an binding of probes by surface of larval dorsal protegulum (asterisks) and epithelial boundary. Dev Genes Evol. 2004;214(4):170–5. borders between protegulum and remaining shell (arrowheads). The con- 6. Monteiro AS, Schierwater B, Dellaporta SL, Holland PWH. A low trol without probes (A). Colorimetric in situ hybridization with sense probe diversity of ANTP class homeobox genes in Placozoa. Evol Dev. of antp (B) and post2 (C) genes, signal developed for the same time as for 2006;8(2):174–82. antisense probes. Fluorescent in situ hybridization with antisense probes 7. Srivastava M, Begovic E, Chapman J, Putnam NH, Hellsten U, of antp (D, E) and post2 (F, G) genes, on E and G combined with DAPI stain- Kawashima T, Kuo A, Mitros T, Salamov A, Carpenter ML, et al. ing of cell nuclei. Dorso-ventral view with anterior to the top (A–D, F) and The Trichoplax genome and the nature of placozoans. Nature. virtual cross section with dorsal to the top (E, G). Dashed lines with letters 2008;454(7207):955–60. on D and F indicate section planes shown on respective plates 8. Yanze N, Spring J, Schmidli C, Schmid V. Conservation of Hox/ParaHox- related genes in the early development of a cnidarian. Dev Biol. 2001;236(1):89–98. Authors’ contributions 9. Finnerty JR. Cnidarians reveal intermediate stages in the evolution of AH designed the study, collected samples and contributed to writing; LG Hox clusters and axial complexity. Am Zool. 2001;41(3):608–20. conducted the in situ hybridizations and the confocal studies, arranged 10. Wallberg A, Thollesson M, Farris JS, Jondelius U. The phylogenetic posi- fgures and drafted the manuscript. Both authors read and approved the fnal tion of the comb jellies (Ctenophora) and the importance of taxonomic manuscript. sampling. Cladistics. 2004;20(6):558–78. 11. Garcia-Fernàndez J. The genesis and evolution of homeobox gene clusters. Nat Rev Genet. 2005;6(12):881–92. Acknowledgements 12. Duboule D. The rise and fall of Hox gene clusters. Development. The staf of UW Friday Harbor Laboratories and crew of the vessel “Centennial” 2007;134(14):2549–60. are gratefully acknowledged for helping in collection of adult T. transversa. We 13. Ferrier DEK, Minguillon C. Evolution of the Hox/ParaHox gene clusters. would like to thank Daniel Thiel, Carmen Andrikou and Chema Martin-Duran Int J Dev Biol. 2003;47(7–8):605–11. for helping with culturing T. transversa larvae and collection of juvenile speci- 14. Seo HC, Edvardsen RB, Maeland AD, Bjordal M, Jensen MF, Hansen mens as well as valuable discussions regarding the project. Additionally, LG is A, Flaat M, Weissenbach J, Lehrach H, Wincker P, et al. Hox cluster very grateful to Aina Børve for her help and assistance during in situ laboratory disintegration with persistent anteroposterior order of expression in procedure. We also would like to thank two anonymous reviewers for their Oikopleura dioica. Nature. 2004;431(7004):67–71. insightful comments, which improved our manuscript. 15. Ikuta T, Yoshida N, Satoh N, Saiga H. Ciona intestinalis Hox gene cluster: Its dispersed structure and residual colinear expression in develop- Competing interests ment. Proc Natl Acad Sci USA. 2004;101(42):15118–23. The authors declare that they have no competing interests. 16. Moreno E, Nadal M, Baguña J, Martínez P. 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